Myosin V is a two-headed unconventional myosin that has an extended neck due to the presence of six light chain binding IQ-repeats. In cells, myosin V is thought to be a vesicle motor. Double-headed (HMM) and single-headed fragments (S1)of mouse myosin V, each tagged with the FLAG-epitope on their carboxyl-terminal end, were expressed in Sf9 cells along with calmodulin. In addition, we purified myosin V from mouse brain and chicken brain. The actin activated MgATPase activity of myosin V purified from brain is strongly activated by the presence of micromolar calcium whereas that of expressed HMM is high even in the absence of calcium suggesting that the tail portion of the molecule has regulatory function. Several lines of evidence suggest that myosin V is a processive motor which can move some distance along actin filaments without dissociation. First, low concentrations of myosin V bound to the surface are able to translocate actin filaments. Second, the kinetics suggest that ADP release is partially or fully rate limiting and that the steady state intermediate is a strongly bound state. Third, single molecules are able to generate multiple steps in vitro using an optical trap. Fourth, electron microscopic data demonstrates that both heads of myosin V can bind to the same actin filament in the presence of ATP. The distance between the heads corresponds to 13 actin monomers (36 nm) or one helical repeat. Consistent with these data the optical trapping results show that the distance between stepping events in a run is 36 nm. In contrast, the first step in a run or the step size of a single attachment event is only 25 nm. This suggests that the second head must be able to search out the appropriate actin monomer while being tethered by the attached head. The power stroke takes place in two unequal steps. Measurement of the stiffness of the attached myosin V heads during a processive runs shows that periods of reduced stiffness can be detected just before stepping events. This probably represents transient periods of single-headed attachment. Electron microscopy of myosin heads in the presence or absence of ATP not attached to actin reveals distinctive differences in orientations of the motor domain compared to the lever arm suggesting that we're able to see the power stroke even in the absence of actin.